Methylation of CpG islands is an epigenetic mechanism of regulating gene expression that has been described in great detail for colon cancer. Methylation is an important mechanism of gene silencing that can suppress the expression of tumor suppressor genes and other growth regulatory genes important in neoplasia. We hypothesize that methylation is an important mechanism of gene silencing in a subset of endometrial cancers. Endometrial cancer is a complex, heterogeneous disease that can be broadly classified into two groups according to histopathological, molecular, and clinical characteristics. Type 1 endometrial cancer is generally associated with estrogen exposure and is clinically less aggressive. Type 2 cancers are not dependent on estrogen and are associated with local recurrence, distant metastasis, and shortened survival. There are numerous patient populations at risk for developing endometrial cancer. High risk characteristics include endometrial hyperplasia, anovulatory cycles, tamoxifen use, obesity with insulin-resistance, and exposure to environmental estrogens. For the first three specific aims, a large panel of markers known to be methylated in various cancers will be used to generate molecular fingerprints of endometrial tissues. Aim 1 will examine CpG island methylation in Type 1 and Type 2 endometrial cancers to support the hypothesis that methylation profiling can provide molecular clues to the differences between Type 1 and Type 2 cancers. Aim 2 will use methylation profiling of endometrial hyperplasia, a precursor to endometrial cancer, to determine if CpG island methylation is an early event. Aim 3 will determine if methylation profiling can accurately identify "at-risk" endometrium. For these experiments, benign endometrium from women with anovulatory cycles or from women taking tamoxifen for breast cancer treatment/prevention will be examined. Both of these groups are at increased risk for developing endometrial cancer. This aim will allow us to explore the feasibility of using CpG island methylation as a tissue biomarker of endometrial cancer risk. Aim 4 will clone genes differentially methylated in endometrial cancer. This aim will allow for the discovery of novel markers of methylation that can potentially be used as therapeutic targets or tissue prognostic indicators. These aims, coupled with the Q-PCR and eDNA array analyses of Project 4, will help construct detailed molecular profiles of endometrial cancer and endometrium at risk for progression to cancer.